P898 StaphPlex System for rapid and simultaneous identification, antibiotic resistance determination and Panton-Valentine leukocidin detection of staphylococci from positive blood cultures

Molecular diagnostics for staphylococci P897 Expression of icaA and icaD genes by quantitative real-time PCR in correlation to biofilm synthesis among methicillin-resistant coagulase-negative staphylococci A. Foka, V. Chini, E. Petinaki, G. Dimitracopoulos, I. Spiliopoulou (Patras, GR) Objectives: Among pathogens causing hospital infections methicillinresistant coagulase–negative staphylococci (MR-CNS) have become predominant, especially in neonatal intensive care units (nICU). This results their ability to form biofilm, especially among patients with invasive procedures. The purpose of the present study was to investigate slime production in correlation to the presence and expression of ica operon among MR-CNS isolated from different patients hospitalised in a nICU. Methods: During a two-year period 132 (MR-CNS) were isolated from different inpatients at the nICU. Biofilm production was detected by the qualitative and quantitative methods. PCR was performed for the detection of the four genes of the ica operon, (icaA, icaD, icaB, icaC) and of the insertion sequence element IS256. Fourteen representative strains, 11 slime-positive and three slime-negative, were selected for reverse transcription quantitative relative Real time PCR (qRT-PCR) for the detection of the expression levels of icaA and icaD genes. Total RNA was isolated by the Trizol method, and a part of the 23S rRNA was used as the reference gene in the relative quantification of ica genes using the SYBR® Green I chemistry; results were calculated by the Pfaffl method. Results: Both methods for bioflilm formation revealed that 117 (89%) isolates were biofilm-positive. PCR showed a variation of possession of ica genes. The selected 14 strains were positive for icaA and icaD by PCR screening. Seven out of the 11 slime-positive strains carried also IS256. Among them, two showed high, two medium and three strains low expression levels of both ica genes by qRT-PCR. In the remaining four slime-positive strains that did not carry IS256, two showed medium and two low expression levels of both ica genes. Two out of the three slimenegative MR-CNS possessed IS256 and all three had low expression levels of ica genes. Conclusions: The majority of MR-CNS associated with infections in the nICU produced biofilm. The insertion sequence element IS256 does not always inactivate the expression of ica operon. Other factors are involved in biofilm formation. P898 StaphPlex System for rapid and simultaneous identification, antibiotic resistance determination and Panton-Valentine leukocidin detection of staphylococci from positive blood cultures Y.W. Tang, A. Kilic, Q. Yang, H.J. Li, R.S. Miller, M. McCormac, K.D. Tracy, C.W. Stratton, J. Han (Nashville, Huntsville, US) Objectives: Current phenotypic methods take several days for identification and antimicrobial susceptibility testing of staphylococcal isolates after Gram-positive cocci in clusters (GPCC) are seen in positive blood cultures. We developed and validated a StaphPlex system that amplifies and detects 18 gene targets simultaneously in one reaction for identification, Panton-Valentine leukocidin (PVL) gene detection, and antimicrobial resistance determination of staphylococci. Methods: We collected positive blood culture specimens in which GPCC were seen and compared the results of the StaphPlex System with staphylococcal identification and antimicrobial resistance determination obtained by phenotypic methods. Results: Among a total of 360 GPCC specimens, 265 (73.6%), 41 (11.4%), 32 (8.9%), 11 (3.1%), and 11 (3.1%) were identified as coagulase-negative Staphylococci (CoNS), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus (MSSA), mixed infections of CoNS and MRSA, and non-staphylococci, respectively. The total agreement rate was 91.9% in comparison with those identified by conventional phenotypic methods. The 277 CoNS specimens were further speciated to 205 (74.0%) Staphylococcus epidermidis, 9 (3.3%) Staphylococcus haemolyticus, 25 (9.0%) Staphylococcus

S233 hominis, 1 (0.4%) Staphylococcus lugdunensis, and 37 (13.4%) other CoNS with an accordance rate of 84.6% when compared to an API STAPH identification. High specificity and low sensitivity were noticed when aacA, ermA, ermC, tetM and tetK genes were detected to predict in vitro antimicrobial susceptibilities. The StaphPlex system presented a sensitivity of 100.0% and specificity ranged from 95.5% to 100.0% when used for staphylococcal cassette chromosome mec typing and PVL gene detection. Conclusion: StaphPlex provides accurate and relevant staphylococcal identification, PVL gene detection, and antimicrobial resistance determination within five hours, which significantly shortens the time usually needed for phenotypic identification and antimicrobial susceptibility testing. P899 Comparison of enterotoxin genes in Staphylococcus aureus from nasal carriage and bacteraemia J.Y. Baek, K.S. Ko, K.R. Peck, J.H. Song (Seoul, KR) Background and Objective: The prevalence of enterotoxin genes within S. aureus isolated in Korea including both in community-setting and in hospital-setting has not been reported. Material and Methods: We analyzed 95 S. aureus colonisers (18 MRSA and 77 MSSA) isolated from children attending outpatient clinics and 70 S. aureus isolates (36 MRSA and 34 MSSA) from patients with bacteraemia. spa typing, multilocus sequence typing, and SCCmec typing were performed to characterise S. aureus isolates. Enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sek, and tst) and pvl gene were assayed by method of PCR. Results: The most common enterotoxin genes were seg, sei, and tst in both nasal carriage strains and S. aureus from bacteraemia (60.0% and 58.6% for seg, 69.5% and 55.7% for sei, and 52.6% and 42.9% for tst, respectively). Possession rate of enterotoxin genes was not significantly different between isolates from nasal carriage and bacteraemia, except for sec and seh genes. While only two isolates of nasal carriage (2.1%) were carrying sec gene, 34.3% of isolates from bacteraemia were positive. Interestingly, sec gene was found only in MRSA isolates. For seh gene, it was positive in 30.5% of nasal carriage strains, but only 8.6% of bacteraemia strains possessed it. No isolates contain pvl gene. While the most prevalent clone was ST30 (spa motif, WG/FKAOMQ) (34/95 isolates, 35.8%) in nasal carriage, ST5 (spa motif, DMGMK) was the most frequently found clone in bacteraemia strains (24/70 isolates, 34.3%). Bacteraemia strains belonging to the same clone have the similar enterotoxin gene pattern, but it did not in nasal carriage strains. Conclusion: S. aureus isolates from bacteraemia and nasal carriage showed different genetic backgrounds, that is, no close relation each other. There was no evidence that S. aureus isolates from bacteraemia possessed more enterotoxins genes than nasal carriage. P900 Performance of the GeneXpert® system for detection of methicillin-resistant Staphylococcus aureus including the non-typeable clone from animal origin M.W.M. Wassenberg, S. Nijssen, X.W. Huijsdens, A.M.C. Bergmans, C.M.J.E. Vandenbroucke-Grauls, M.J.M. Bonten, J.A.J.W. Kluytmans (Utrecht, Breda, Bilthoven, Amsterdam, NL) Objectives: The rapid and accurate identification of methicillin-resistant Staphylococcus aureus (MRSA) is of great importance to control the spread of MRSA. Since 2003 a MRSA clone from animal origin is observed with rapidly increasing prevalence in the Netherlands and this isolate is non-typable (NT) with SmaI PFGE. The recently developed GeneXpert® system allows molecular detection of MRSA with a simple procedure in 75 minutes. The objective of this study was to assess the in vitro sensitivity of the Xpert MRSA Assay performed in the GeneXpert® system to detect MRSA including NT-MRSA isolates. Methods: A collection consisting of 35 NT-MRSA and 73 typable MRSA strains isolated from humans in the period January 2003 to